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WO2005068655A2 - Procedes d'evaluation d'une reponse inflammatoire tissulaire au moyen de profils d'expression de cellules endotheliales - Google Patents

Procedes d'evaluation d'une reponse inflammatoire tissulaire au moyen de profils d'expression de cellules endotheliales Download PDF

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WO2005068655A2
WO2005068655A2 PCT/GB2005/000057 GB2005000057W WO2005068655A2 WO 2005068655 A2 WO2005068655 A2 WO 2005068655A2 GB 2005000057 W GB2005000057 W GB 2005000057W WO 2005068655 A2 WO2005068655 A2 WO 2005068655A2
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u95av2
transcripts
protein
inflammatory response
tissue
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WO2005068655A3 (fr
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Steven Kevin Smith
David Stephen Charnock-Jones
Cristin Gregor Print
Nicola Anne Johnson
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Cambridge University Technical Services Ltd CUTS
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Cambridge University Technical Services Ltd CUTS
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Priority to EP05701827A priority Critical patent/EP1711630A2/fr
Priority to GB0615106A priority patent/GB2424947A/en
Priority to AU2005205218A priority patent/AU2005205218A1/en
Priority to CA002551677A priority patent/CA2551677A1/fr
Priority to JP2006548388A priority patent/JP2007522800A/ja
Publication of WO2005068655A2 publication Critical patent/WO2005068655A2/fr
Publication of WO2005068655A3 publication Critical patent/WO2005068655A3/fr
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Definitions

  • the present invention relates to gene expression profiles of endothelial cells and to the use of these profiles in the assessment of disease conditions and in therapy.
  • leucocytes The migration of leucocytes into tissues is a key part of the normal function of the immune system, mucosal surfaces, the uterine endometrium, mammary gland and ovary.
  • emigration of leucocytes from the intravascular space into the tissues can also be stimulated by the inflammation response.
  • Inflammation is a response of the body to damage or infection, and typically involves movement of leucocytes and proteins across endothelial walls and into tissues.
  • the inflammation response involves a number of signalling molecules. Some of these act on the endothelium to cause the endothelial cells to adhere less tightly to one another and/or to make their surfaces adhesive to passing white blood cells. Other signals act as chemoattractants for the leucocytes, or promote expression of adhesion molecules on the leucocytes. Yet other signals regulate leucocyte activation and survival.
  • Signalling molecules include quick release' mediators that are involved in increased vascular permeability, such as histamine. These act synergistically with the later stage ⁇ process induced' mediators. These later stage mediators include chemokines, which attract leukocytes into areas of inflammation, and cytokines, subsequently released from inflammatory cells and activated endothelial cells. TNF- ⁇ , IL-l ⁇ and IL-8 are inflammatory mediators found at high concentrations at sites of inflammation, and endothelial cells possess receptors for these mediators (1-3) .
  • TNF- ⁇ is a prototypical cytokine, which was originally isolated from macrophages, but is now known to be generated by a variety of cells, including lymphocytes, monocytes, neutrophils, T-cells, myocytes and smooth muscle cells.
  • TNF receptor 1 engagement by TNF- ⁇ leads to the initiation of apoptosis in a wide variety of cell types. Receptor binding also results in sustained NF- ⁇ B activation, required for expression of a repertoire of genes involved not only in the inflammatory response, but also increased cell survival and enhanced resistance to TNF- ⁇ -induced apoptosis.
  • TNF- ⁇ phosphatidylinositol
  • SAPK stress activated protein kinases
  • MAPK mitogen activated protein kinases
  • IL-l ⁇ is another prototypical cytokine.
  • the main source of secreted Il-l ⁇ is monocytes, but it is also generated by a wide variety of other cell types, including, notably, activated macrophages, neutrophils, platelets, T- and B-cells, natural killer (NK) cells, endothelial cells, SMCs (smooth muscle cells) and fibroblasts.
  • IL-l ⁇ is synthesised as a precursor and, once proteolytically cleaved, can activate many cell types with roles in immunity and inflammation.
  • TNF- ⁇ IL-l ⁇ activates the NF- ⁇ B (nuclear factor KB) survival pathway, as well as MAPK cascades p42/p44, p38 and Jnk (5) .
  • IL-8 is a chemokine that has been closely linked with angiogenesis .
  • the main sources of IL-8 are monocytes, neutrophils, T-cells, NK cells, endothelial cells, fibroblasts and epithelial cells.
  • IL-8 is involved in the process of leukocyte transmigration into tissues, as well as other aspects of the inflammatory response. Its production is not constitutive but is induced by pro-inflammatory cytokines.
  • IL-8 binds to heparin and induces endothelial cell migration, proliferation and survival in vitro ( ⁇ ) .
  • IL-8 and its receptors appear to be critical in angiogenesis and tumour progression (7, 8).
  • mediators of inflammation such as TNF- ⁇ , IL-1 and IL-8 act on target cells within inflamed tissues such as endothelial cells, fibroblasts and leukocytes.
  • the effects of combinations of inflammatory mediators such as these three is synergistic - i.e. the action of these factors ⁇ in concert' is likely to be different from the sum of their individual effects .
  • the inflammatory response of tissues typically involves the abnormal or excessive movement of leucocytes and proteins into the tissue. This can result in a large number of acute or chronic inflammation disorders.
  • Leucocyte and protein movement can also play a role in conditions which are not primarily inflammatory diseases, but in which the tissues demonstrate an inflammatory response.
  • the diseases of atherosclerosis and endometriosis may rely on the migration of leucocytes into the lesions. Repair of tissue necrosis that occurs in stroke or heart attack also requires inflammation responses.
  • Tumour growth may be regulated by leucocytes that migrate from the blood into the tumour. These leucocytes release factors that directly promote tumour cell growth and tumour angiogenesis.
  • leucocytes that migrate into tumours mediate an immune responses towards tumour cell- specific antigens with subsequent anti-tumour effect.
  • the role of inflammation in tumour growth is therefore complex, and involves a balance between stimulatory and inhibitory pathways .
  • inflammation responses are essential features of physiological processes such as female fertility and wound healing. There is a need for methods to modulate inflammatory responses in order to modulate these processes.
  • HUVEC human coronary artery endothelial cells
  • UtMVEC human uterine microvascular endothelial cells
  • the present invention provides a means for assessment of (e.g., diagnosis, prognosis or monitoring of) a tissue inflammatory response or a condition associated therewith.
  • a tissue inflammatory response is typically associated with one of a number of conditions and it may also involve the migration of leucocytes across an endothelium. Therefore, by determining a tissue inflammatory response, a determination may also be made of the condition with which that response may be associated. Also, the finding that a number of transcripts are regulated in these responses and hence conditions, both of genes known as such and from ESTs, allows the provision of novel assay targets and the development of new therapies for such conditions.
  • the present invention provides a method of assessing a tissue inflammatory response, comprising: making a quantitative determination of the level of at least five transcripts shown in Table 1, or proteins encoded thereby, in a sample; and comparing the abundance of said transcripts or proteins so determined with the level of said transcripts or proteins obtained from a control sample of cells.
  • the sample comprises cells obtained from a site within a patient believed to be affected by a tissue inflammatory response, suitably endothelial cells, or patient blood, serum or urine.
  • a tissue inflammatory response suitably endothelial cells, or patient blood, serum or urine.
  • the invention provides a gene chip array suitable for use in the above-described method of the invention, comprising at least five nucleic acids suitable for detection of at least five transcripts shown in Table 1; optionally a control specific for said transcripts; and optionally at least one control for the gene chip.
  • the invention provides a protein based assay suitable for use in the above-described method of the invention, for the assessment of at least five proteins encoded by transcripts including those shown in Table 1; optionally a control specific for said proteins; and optionally at least one control for the assay.
  • the invention provides an assay method for determining a modulator of a tissue inflammatory response or a condition associated therewith, wherein said method comprises a) providing a protein encoded by transcripts from Table 1; b) bringing the protein into contact with a candidate modulator of its activity; c) determining whether said candidate modulator is capable of modulating the activity of the protein;
  • said method comprises; a) providing an endothelial cell in culture; b) bringing said cell into contact with a candidate modulator of said tissue inflammatory response; and c) determining whether said candidate modulator is capable of modulating the level of at least one transcript selected from the transcripts of Table 1.
  • Modulators obtained by such methods may be used in a method of modulating tissue inflammatory responses in a human or animal subject, e.g., so as to treat a condition discussed below.
  • the identification of genes of previously unknown function and of ESTs has allowed new potential targets for therapeutic intervention to be developed.
  • the invention provides a vector comprising a sequence encoding a transcript from Table lb operably linked to a promoter for the transcription of said sequence.
  • Such vectors are useful in the expression of proteins which may be therapeutics or therapeutic targets.
  • the vectors may also have direct therapeutic use in themselves, e.g., in gene therapy applications .
  • Table la lists transcripts of genes whose levels are regulated in endothelial cells by inflammatory mediators TNF ⁇ , IL-l ⁇ , and IL-8, where the genes have a previously ascribed function.
  • Table lb lists transcripts of genes or ESTs whose levels are regulated in endothelial cells by inflammatory mediators TNF ⁇ , IL-l ⁇ and IL-8, where the genes/ESTs do not have a previously ascribed function.
  • Table 2a provides nucleic acid sequences for each of the probes mentioned in Table la.
  • Table 2b provides nucleic acid sequences for each of the probes mentioned in Table lb.
  • Table 1 and 2 is to be construed as meaning Tables la or lb together, or Tables 2a and 2b together, respectively, unless the context is explicit to only one of these component parts of Table 1 or Table 2 respectively.
  • the present invention is concerned with a method for the assessment of a tissue inflammatory response. It is generally recognised, as is discussed above, that • leucocytes are involved in the inflammatory responses of tissues.
  • An inflammatory response may involve the movement of leucocytes across an endothelium.
  • proteins which are involved in such an inflammatory response may also cause the movement of leucocytes to the inflamed tissue, may participate in or be responsible for the activation of the leucocytes, the activation and angiogenesis of endothelial cells, or may influence the inflammatory response by inhibiting the apoptosis of leucocytes or endothelial cells.
  • a tissue inflammatory response is typically involved with a condition or disease state of the patient.
  • condition as used herein is meant any disease state or body response which involves, causes or is associated with the tissue inflammatory response.
  • the tissue inflammatory response may either be excessive or unwanted, for example as may be the case in an inflammatory disease such as those discussed herein below, or may be insufficient, such as, for example in conditions associated with defective adhesion proteins, such as leucocyte adhesion deficiency types 1 and 2 (LAD-1 and LAD-2) .
  • Tissue inflammatory responses and trans-endothelial leucocyte movement can be affected by changes in the endothelial cells, for example by changes in adhesion molecule expression, which alters the ability of the leucocytes to adhere to the endothelial cells, or alters the chemokine-mediated recruitment of leucocytes to the inflammed tissue.
  • the changes in the endothelial cells are in response to an inflammatory signal .
  • a condition to be assessed by the method of the invention may be one in which endothelial cells in the location of the inflammatory response or affected by the inflammatory response are altered so as to promote an inflammatory process and allow the movement of leucocytes across that endothelium.
  • a tissue inflammatory response to be assessed by the method of the invention is one in which inflammatory signals are issued.
  • inflammatory diseases e.g., vasculitic syndromes, atherosclerosis and associated diseases, conditions involving tumour growth and chronic transplant rejection.
  • Inflammatory diseases include inflammatory bowel disorders, psoriasis, ischemic reperfusion, adult respiratory distress syndrome, asthma, allergic rhinitis, dermatitis, meningitis, encephalitis, uveitis, diseases involving leucocyte diapedesis, central nervous system inflammatory disorders, Alzheimer's, endometriosis, multiple sclerosis, multiple organ injury syndrome, alcoholic hepatitis, bacterial pneumonia, antigen-antibody complex mediated diseases; inflammation of the lung (including pleurisy, alveolitis, pneumonia, chronic bronchitis, bronchiectasis, cystic fibrosis and COPD) , vasculitis, polyarteritis nodosa, giant cell arteritis, microscopic polyarteritis, pre-eclampsia and autoimmune diseases (such as rheumatoid arthritis, Sjorgen's syndrome, and the multiple forms of renal failure which are primarily due to autoimmune attack directed at
  • leucocyte migration across the endothelial layer into blood vessel walls at the site of an atherosclerotic plaque is driven by inflammatory signals from leucocytes associated with the plaque and the underlying endothelial cells.
  • Plaque instability and subsequent rupture leading to thrombosis and vessel stenosis or occlusion may be associated with inflammatory effects on the endothelial cells within the area of the plaque.
  • Tumour growth may be regulated by leucocytes that migrate from the blood into the tumour, and which release factors that directly promote tumour cell growth and tumour angiogenesis.
  • cancers and especially those involving solid tumours may also be a relevant therapeutic indication.
  • Chronic transplant rejection critically involves immune cell migration across blood vessels and immune attack directed towards endothelial cells and hence is suitable for assessment by the method of the invention.
  • bodily fluids such as blood, serum and urine may be collected to reveal inflammation-induced changes within these fluids. Such changes may be demonstrated by, for example, an alteration in the sample in the abundance of endothelial cell-derived proteins that are encoded by the sequences in table 1.
  • the present invention provides a method for the diagnosis of a condition with which a tissue inflammatory response is associated comprising the determination of the abundance of endothelial cell-derived proteins encoded by at least five of the transcripts of Table 1 in a sample from a patient suspected of suffering from such a condition.
  • the data obtained in the present invention shows that contacting endothelial cells with TNF ⁇ , IL-l ⁇ and IL-8 results in characteristic changes to certain transcripts, taking into account variation between individuals and between different endothelial tissues. These changes may take the form either of a pattern of response or may affect individual transcripts. Accordingly, it is possible to use these characteristic changes as a reference for the diagnosis of conditions such as those discussed above.
  • assessment of the condition may be a determination of the severity or precise clinical sub-type of the condition. It may also be a method of prognosis of the future course of the condition. Furthermore assessment can be over a period of time, for example in the monitoring of the treatment of a condition.
  • the present invention provides a method of assessing a tissue inflammatory response by comparing the transcript levels in a sample taken from a patient showing an inflammatory response with the transcript levels of the same genes in a control sample.
  • the sample may be either of cells from a specific tissue demonstrating an inflammatory response, such as endothelial cells, or may alternatively be of a protein- containing bodily fluid, such as blood, serum or urine.
  • a control sample for the above methods is typically a sample of unaffected cells, that is cells from a tissue not demonstrating a tissue inflammatory response or cells believed not to be affected by the condition. It is preferred that the test and control sample are both of the same cell type and more preferred that the test and control sample are of endothelial cells. Most preferably, the test and control sample are of the same endothelial cell type, i.e. from the same tissue location. If required, the control sample may be taken from a population of subjects. Where a population of subjects is used, the comparison may be made with the average (e.g., mean or median) transcript level in samples of cells from said population. Affected tissue may also be used as a control.
  • the affected tissue will be from a patient other than that from which the test sample was taken. It is generally preferred that, if affected tissue is being used as a control, the patient from which that sample is removed has or is believed to have the same condition as the patient from which the test sample is removed.
  • the method of the invention may comprise the use of more than one control; for example the sample to be tested may be compared to a normal or unaffected sample from the same patient and the transcript level of an affected sample from another patient or patients.
  • the method of the invention allows for the monitoring of a condition, either before, during or after treatment.
  • a sample is also obtained from the patient at an earlier time point, so as to provide a historical record.
  • the method allows for assessment of the effectiveness of a particular treatment.
  • the invention is performed by looking at the level of a plurality of gene transcripts or plurality of proteins derived from these transcripts. This is because we have found that between individual subjects, the transcript level of individual genes may vary. It is therefore desirable that the transcript level is assessed for several genes. For example, it may be assessed for at least
  • transcripts of Table 1 preferably at least 10 and more preferably at least 20 transcripts or proteins derived from transcripts of Table 1.
  • the level of expression may be determined either for the genes or proteins individually or in specific combinations, or a pattern of expression of a plurality of genes may be determined.
  • the level of the transcripts assessed is compared in a test and a control sample. It may be that the response to an inflammatory signal causes the level of the transcripts either to rise or to fall. Hence there may be either up-regulation of the transcripts in response to an inflammatory signal or down-regulation. It is not important to the present invention whether there is either up-regulation or down-regulation, but rather that there is change from the control.
  • the specific amount of up- or down-regulation may be determined. This can be expressed as a "fold" increase or decrease. It is not important to the present invention how large is the increase or decrease in transcript level, but rather it is important that there is a significant change from the control. In the present invention it is generally preferred that there be at least a two-fold change, i.e. increase or decrease, in the transcript level. There is no upper limit on the amount of increase or decrease in transcript or protein level, but we have found that a 50-fold change in the level of certain transcripts and apparent e novo upregulation of certain proteins can be demonstrated.
  • transcripts or proteins are assessed to be regulated by the same amount. Of more significance in this respect is the establishment of a pattern of regulation.
  • the transcripts or proteins assessed may include one or from transcripts from one or more families or groups of protein.
  • the method involves determining the levels of at least one transcript or protein in two or more of such families or groups of protein, preferably three, four, five or more, and more preferably each of such families or groups of protein.
  • Typical such groups or families include proteases, bone morphogenetic proteins, cell signalling proteins, proteins involved in transcriptional regulation, enzymes, cell cycle regulation proteins, cell communication proteins, nuclear proteins, complement proteins, extracellular matrix proteins, proteosomes, heavy metal binding proteins, TNF-receptor superfamily proteins, HLA proteins, cell adhesion proteins, cytokines/chemokines, cytokine/chemokine receptors, cytoskeletal proteins, regulators of apoptosis, growth factors and growth factor receptors.
  • proteases include proteases, bone morphogenetic proteins, cell signalling proteins, proteins involved in transcriptional regulation, enzymes, cell cycle regulation proteins, cell communication proteins, nuclear proteins, complement proteins, extracellular matrix proteins, proteosomes, heavy metal binding proteins, TNF-receptor superfamily proteins, HLA proteins, cell adhesion proteins, cytokines/chemokines, cytokine/chemokine receptors, cytoskeletal proteins, regulators of apoptosis, growth factors and growth factor receptors.
  • transcripts or proteins assessed include one or more selected from cytokines/- chemokines, cytokine/chemokine receptors, cytoskeletal proteins, extracellular matrix protins, regulators of apoptosis, growth factors and growth factor receptors.
  • transcripts or proteins assessed include one or more of: colony stimulating factor 3 (granulocyte) , colony stimulating factor 2 (granulocyte- macrophage) , granulocyte chemotactic protein 2, diubiquitin, ELAM-1, TNF-induced protein 6, Exodus 1, IL-l ⁇ , VCAM-1, ICAM- 1, IAP1, TNF-inducible A20, RIPK2, MMP 10, TRAF1, JAK binding protein, dual specificity phosphatase 4, IL-6, IL-8, Gro- gamma, MCP-1, Gro-beta, Gro-alpha, ENA-78, fractalkine, small inducible cytokine subfamily A14, small inducible cytokine A5, LI7 receptor, toll/interleukin 1 receptor-like 4, CCAAT,
  • the transcripts or proteins assessed include one or more of: colony stimulating factor 3 (granulocyte) , colony stimulating factor 2 (granulocyte-macrophage) , granulocyte chemotactic protein 2, diubiquitin, ELAM-1, TNF-induced protein 6, Exodus 1 and IL-l ⁇ .
  • the method involves determining a pattern of expression of transcripts, wherein the transcripts include at least some of the transcripts illustrated in Table 1 herein.
  • the transcript or protein level of a gene or genes may be determined by any suitable means. Where many different gene transcripts are being examined, a convenient method is by hybridisation of the sample (either directly or after generation of cDNA or cRNA) to a gene chip array.
  • genes are all present in commercially available chips from Affymetrix, and these chips may be used in accordance with protocols from the manufacturer.
  • methods for the provision of microarrays and their use may also be found in, for example, WO84/01031, WO88/1058, WO89/01157, W093/8472, W095/18376/ W095/18377, W095/24649 and EP-A-0373203 and reference may also be made to this and other literature in the art.
  • Table 1 provides the names of genes and these may be used to obtain their DNA sequences and encoded protein sequences from databases such as Genbank.
  • the particular sequences used on the Affymetrix chip we have used may be determined by the Affymetrix reference numbers supplied in the Table, which are publicly available and may be related directly to Genbank reference numbers.
  • EST gene sequences in some cases are also given by Genbank reference numbers. Those of skill in the art may refer to either of the Affymetrix reference number or the Genbank reference number in practicing the present invention.
  • Table 2 provides cDNA sequence information for each of the probes used on the Affymetrix chip that are also listed in Table 1.
  • Table 1 lists the probe set used to obtain the specific protein/EST listed therein and Table 2 provides the cDNA sequence for each probe set mentioned in Table 1.
  • n represents in each case a region of the nucleic acid that was not probed by the probe set sequences on the gene chips.
  • n represents in each case a region of the nucleic acid that was not probed by the probe set sequences on the gene chips.
  • PCR methods may be used, e.g. based upon the ABI TaqManTM technology, which is widely used in the art. It is described in a number of prior art publications, for example reference may be made to WO00/05409. PCR methods require a primer pair which target opposite strands of the target gene at a suitable distance apart (typically 50 to 300 bases) . Suitable target sequences for the primers may be determined by reference to Genbank sequences as mentioned above.
  • the invention provides a gene chip array comprising at least five nucleic acids suitable for detection of at least five transcripts shown in Table 1 (either directly or after generation of cDNA or cRNA) ; optionally a control specific for said transcripts; and optionally at least one control for said gene chip.
  • the invention provides a gene chip array for detection of at least five transcripts shown in Table 1 comprising at least five nucleic acids selected from Table 2 capable of detecting the presence of said at least five transcripts; optionally a control specific for said transcripts and optionally at least one control for said gene chip.
  • the gene chip array comprises at least five different nucleic acids, preferably at least 10 or 20 different nucleic acids.
  • the number of sequences in the array will be such that where the number of nucleic acids suitable for detection of the Table 1 transcripts (or the number of Table 2 sequences) is n, the number of control nucleic acids specific for individual transcripts is n' , where n' is from 0 to 2n, and the number of control nucleic acids (e.g.
  • n + n' + m represent at least 50%, preferably 75% and more preferably at least 90% of the nucleic acids on said chip.
  • the provision of the transcripts and proteins encoded thereby in Table 1 allows for the production of appropriate tailored protein chips.
  • Such chips may be used in the form of an antibody chip, or may encompass multi-target ELISA, or proteomic analysis.
  • methods for the provision of protein assays and their use may be found in, for example, U.S. Patent 6,329,209 and U.S. Patent No. 2,656,508. Such methods are common within the art.
  • the present invention provides a protein based-assay suitable for use in the above- described method of the invention, for the assessment of at least five proteins encoded by transcripts including those shown in Table 1; optionally a control specific for said proteins; and optionally at least one control for the assay.
  • the protein-based assay is an antibody chip or ELISA, preferably multi-target ELISA.
  • Assay methods of the present invention may be practiced in a wide variety of formats, for example on protein or nucleic acid components or in whole cells in culture.
  • the present invention provides in its second aspect an assay method for a modulator of a tissue inflammatory response or a condition associated therewith, comprising: (a) providing a protein encoded by a transcript of Table l; (b) bringing said protein into contact with a candidate modulator of its activity; and (c) determining whether said candidate modulator is capable of modulating the activity of said protein.
  • the assay is preferably for an activator of the protein, and the assay preferably involves determining whether the modulator is capable of increasing the activity of the protein.
  • the assay may be carried out under conditions where the protein normally shows low or no activity.
  • the assay is preferably for an inhibitor of the activity of the protein, and the assay preferably involves determining whether the modulator is capable of reducing the activity of the protein.
  • the assay may be carried out under conditions in which the protein is normally active.
  • the determination of modulation of activity will depend upon the nature of the protein being assayed.
  • proteins with enzymatic function may be assayed in the presence of a substrate for the enzyme, such that the presence of a modulator capable of modulating the activity results in a faster or slower turnover of substrate.
  • the substrate may be the natural substrate for the enzyme or a synthetic analogue. In either case, the substrate may be labelled with a detectable label to monitor its conversion into a final product.
  • the candidate modulator may be examined for ligand binding function in a manner that leads to antagonism or agonism of the ligand binding property.
  • DNA binding or transcriptional activating activity may be determined, wherein a modulator is able to either enhance or reduce such activity.
  • DNA binding may be determined in a mobility shift assay.
  • the DNA region to which the protein binds may be operably linked to a reporter gene (and additionally, if needed, a promoter region and/or transcription initiation region between said DNA region and reporter gene) , such that transcription of the gene is determined and the modulation of this transcription, when it occurs, can be seen.
  • Suitable reporter genes include, for example, ' chloramphenicol acetyl transferase or more preferably, fluorescent reporter genes such as green fluorescent protein.
  • the present invention provides an assay method for a modulator of a tissue inflammatory response or a condition associated therewith comprising: (a) providing an endothelial cell in culture; (b) bringing said cell into contact with a candidate modulator of said condition; and (c) determining whether said candidate modulator is capable of modulating the level of at least one transcript selected from the transcripts of Table 1.
  • an endothelial cell is cultured. Any appropriate method may be used for the harvesting and culture of the cells. Techniques for the culture of endothelial cells are common in the art.
  • Candidate modulator compounds may be natural or synthetic chemical compounds used in drug screening programmes. Extracts of plants, microbes or other organisms, which contain several characterised or uncharacterised components, may also be used. Combinatorial library technology (including solid phase synthesis and parallel synthesis methodologies) provides an efficient way of testing a potentially vast number of different substances for the ability to modulate an interaction. Such libraries and their use are known in the art for all manner of natural products, small molecules and peptides, among others. Many such libraries are commercially available and sold for drug screening programmes of the type now envisaged by the present invention.
  • a further class of candidate modulator comprises antibodies or binding fragments thereof which bind a protein target.
  • antibody fragments capable of binding an antigen or other binding partner, are the Fab fragment consisting of the VL, VH, Cl and CHI domains; the Fd fragment consisting of the VH and CHI domains; the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; the dAb fragment which consists of a VH domain; isolated CDR regions and F(ab')2 fragments, a bivalent fragment including two Fab fragments linked by a disulphide bridge at the hinge region. Single chain Fv fragments are also included.
  • An antibody specific for a protein may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g.
  • Another class of candidate molecules is peptides, based upon a fragment of the protein sequence to be inhibited.
  • fragments of the protein corresponding to portions of the protein which interact with other proteins or with DNA may be a target for small peptides which act as competitive inhibitors of protein function.
  • Such peptides may be for example from 5 to 20 amino acids in length.
  • the peptides may also provide the basis for design of mimetics.
  • mimetics will be based upon analysis of the peptide to determine the amino acid residues or portions of their side chains essential and important for biological activity to define a pharmacophore followed by modelling of the pharmacophore to design mimetics which retain the essential residues or portions thereof in an appropriate three-dimensional relationship.
  • Various computer-aided techniques exist in the art in order to facilitate the design of such mimetics.
  • Cell based assay methods can be configured to determine expression of the gene either at the level of transcription or at the level of translation. Where transcripts are to be measured, then this may be determined using the methods of the first aspect of the invention described above, e.g. on gene chips, by multiplex PCR, or the like.
  • the assay is preferably for agents which increase the expression of the gene (e.g., by increasing the quantity of the transcript) .
  • agents which increase the expression of the gene e.g., by increasing the quantity of the transcript
  • Such an agent may comprise the coding sequence of the gene itself (i.e., it may be a gene therapy vector) .
  • the assay is preferably for agents which decrease the expression of the gene.
  • Cell based assay methods may be used to screen candidate modulators as described above. They may also be used to screen further classes of candidate modulator, including antisense oligonucleotides .
  • Such oligonucleotides are typically from 12 to 25, e.g. about 15 to 20 nucleotides in length, and may include or consist of modified backbone structures, e.g. methylphosphonate and phosphorothioate backbones, to help stabilise the oligonucleotide.
  • the antisense oligonucleotides may be derived from the coding region of a target gene or be from the 5' or 3' untranslated region.
  • Candidate molecules may further include RNAi, i.e.
  • RNA molecules which are sequence specific for a gene transcript, or a longer RNA sequence which can be processed by the cell into siRNA, and which can be provided to the cell e.g., as a DNA sequence (eliRNA, or expressed long interfering RNA) .
  • They may also include ribozymes which specifically target the transcript mRNA, i.e., a catalytic RNA molecule which cleaves other RNA molecules of a particular nucleic acid sequence. General methods for the construction of ribozymes are known in the art.
  • the assay methods described above are used to determine modulators of activity of a protein or level of a transcript from Table lb.
  • Modulators obtained in accordance with these aspects of the present invention may be used in methods of modulating tissue inflammatory responses and/or conditions associated therewith, e.g., inflammatory conditions, in a patient.
  • the present invention therefore also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a modulator of a tissue inflammatory response and/or a condition associated therewith together with a pharmaceutically acceptable carrier therefor.
  • the modulator will be formulated with one or more pharmaceutically acceptable carriers suitable for a chosen route of administration to a subject.
  • conventional non-toxic solid carriers include, for example, pharmaceutical grades of mannitol, lactose, cellulose, cellulose derivatives, starch, magnesium stearate, sodium saccharin, talcum, glucose, sucrose, magnesium carbonate, and the like may be used.
  • Liquid pharmaceutically administrable compositions can, for example, be prepared by dissolving, dispersing, etc, a modulator and optional pharmaceutical adjuvants in a carrier, such as, for example, water, saline aqueous dextrose, glycerol, ethanol, and the like, to thereby form a solution or suspension.
  • the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents and the like, for example, sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, sorbitan monolaurate, triethanolamine oleate, etc.
  • the composition or formulation to be administered will, in any event, contain a quantity of the active compound (s) in an amount effective to alleviate the symptoms of the subject being treated. Routes of administration may depend upon the precise condition being treated, though since endothelial cells form the lining of the vasculature, administration into the blood stream (e.g. by i.v. injection) is one possible route.
  • the present invention provides a modulator of a tissue inflammatory response and/or a condition associated therewith for use in therapy.
  • the present invention provides the use of a modulator of a tissue inflammatory response in the manufacture of a medicament for the treatment of a condition associated with said response.
  • the sequence encoding a transcript listed in Table lb is operably linked to a control sequence which is capable of providing for expression of the coding sequence by a host cell, i.e., the vector is an expression vector.
  • operably linked refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner.
  • a control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under condition compatible with the control sequences.
  • Suitable host cells include bacteria, eukaryotic cells such as mammalian and yeast, and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, COS cells and many others.
  • the vectors may include other sequences such as promoters or enhancers to drive the expression of the inserted nucleic acid, nucleic acid sequences so that the polypeptide is produced as a fusion and/or nucleic acid encoding secretion signals so that the polypeptide produced in the host cell is secreted from the cell.
  • the vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector.
  • Vectors may further include enhancer sequences, terminator fragments, polyadenylation sequences and other sequences as appropriate.
  • Vectors may be used in vitro, for example for the production of RNA or used to transfect or transform a host cell.
  • the vector may also be adapted to be used in vivo, for example in methods of gene therapy.
  • Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.
  • Vectors include gene therapy vectors, for example vectors based on adenovirus, adeno-associated virus, retrovirus (such as HIV or MLV) or alpha virus vectors.
  • Promoters and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed.
  • yeast promoters include S. cerevisiae GAL4 and ADH promoters, S. pombe nmtl and adh promoter.
  • Mammalian promoters include the metallothionein promoter which is can be included in response to heavy metals such as cadmium.
  • Viral promoters such as the SV40 large T antigen promoter or adenovirus promoters may also be used. All these promoters are readily available in the art.
  • Vectors for production of polypeptides encoded by a transcript of Table lb for use in gene therapy include vectors which carry a mini-gene sequence.
  • Vectors may be transformed into a suitable host cell as described above to provide for expression of a polypeptide of the invention.
  • the invention provides a process for preparing polypeptides encoded by a transcript of Table lb which comprises cultivating a host cell transformed or transfected with an expression vector as described above under conditions to provide for expression by the vector of a coding sequence encoding the polypeptides, and recovering the expressed polypeptides.
  • Polypeptides may also be expressed using in vitro systems, such as reticulocyte lysate .
  • Polypeptides or fragments thereof in substantially isolated form encoded by an EST or gene of Table lb form a further aspect of the present invention. Fragments of the polypeptides will preferably be at least 20 amino acids in size, and preferably from 25 amino acids up to the full length of the polypeptide.
  • a further aspect of the invention is nucleic acid sequences which encode said polypeptides and fragments thereof.
  • Such nucleic acid sequences may be included in vectors such as those described above.
  • a gene or EST of Table lb may be linked in-frame to a translational initiation region for translation of said sequence, or alternatively it may be in an anti-sense orientation for transcription of anti-sense RNA.
  • TNFa, IL-l ⁇ and Interleukin-8 regulate transcript abundance To allow a study of the effect of TNF ⁇ , IL-l ⁇ and Interleukin- 8 on transcript abundance, seven independent primary cultures of HUVEC were cultured with a mixture of 15ng/mL of each of TNF- ⁇ , Interleukin-l ⁇ and Interleukin 8 for 24 hours. The RNAs extracted from these cultures were then used to prepare complex cRNA probes, which were hybridised to an Affymetrix U95A genechip (a 12, 600-element Affymetrix gene array chip).
  • Affymetrix U95A genechip a 12, 600-element Affymetrix gene array chip.
  • Transcript abundance data were globally scaled to bring the median gene expression of each chip (excluding control genes) to 1.
  • the "LOESS" function of the "R” statistical software system was applied to the log transformed average difference values of each array in comparison to a control array.
  • the control array chosen was that which bore the closest similarity to the other six controls by Euclidean distance.
  • HCAEC human coronary artery endothelial cells
  • UtMVEC human uterine microvascular endothelial cells
  • transcripts which are regulated in all three cell types are believed to be particularly useful in the various methods of the present invention, since they are likely to be meaningful in a variety of inflammatory conditions affecting distinct organs.
  • transcripts which are modulated by inflammatory signals in all three endothelial cell types tend also to be strongly modulated by inflammatory signals in HUVEC.
  • Table la and lb show a consensus set of transcripts. These were selected on the basis that their level before and after treatment with inflammatory mediators differs by a factor of two or more (in either direction) , with a Baysian P value of less than 0.001. These have been grouped according to gene family.
  • Table 1 shows the extent of regulation in HUVEC cells, and also in HCAEC and UtMVEC cells.
  • HUVECs isolated from seven different individuals were cultured in Microvascular Endothelial Cell Growth Medium-2 (EBM-MV, Biowhittaker) .
  • Medium was supplemented with a proprietary mixture of human Epidermal Growth Factor, Hydrocortisone, Vascular Endothelial Growth Factor, human Fibroblast Growth Factor, human recombinant Insulin-like Growth Factor, Ascorbic Acid, Gentamicin, Amphotericin-B and FBS (5%) (Microvascular BulletkitTM reagents, Biowhittaker.
  • EBM-MV Microvascular Endothelial Cell Growth Medium-2
  • Single donor-derived human coronary artery ECs and uterine myometrial microvascular ECs were purchased from Biowhittaker. Cells were cultured and treated under identical conditions as HUVECs
  • Biotinylated target cRNA was prepared according to Affymetrix protocol, hybridised to Affymetrix human U95Av2 chips and globally scaled using Microarray Suite Version 4.0 or 5.0 software.

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Abstract

L'invention concerne des procédés d'évaluation d'une réponse inflammatoire tissulaire, qui consistent à effectuer une détermination quantitative du niveau d'au moins cinq transcrits indiqués dans le Tableau 1, ou des protéines codées par ceux-ci, dans un échantillon; et à comparer l'abondance de ces transcrits ou protéines ainsi déterminés au niveau du transcrit obtenu de l'échantillon témoin. L'invention concerne en outre des procédés de diagnostic d'un état associé à une réponse inflammatoire tissulaire, ainsi que des réseaux de puces à ADN et des analyses à base de protéines que l'on peut utiliser dans ces procédés. L'invention concerne enfin des procédés d'analyse permettant de déterminer un modulateur d'une réponse inflammatoire tissulaire ou d'un état qui lui est associé.
PCT/GB2005/000057 2004-01-16 2005-01-14 Procedes d'evaluation d'une reponse inflammatoire tissulaire au moyen de profils d'expression de cellules endotheliales Ceased WO2005068655A2 (fr)

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AU2005205218A1 (en) 2005-07-28
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GB0615106D0 (en) 2006-09-06
WO2005068655A3 (fr) 2005-11-17
CN1934274A (zh) 2007-03-21
GB0400976D0 (en) 2004-02-18
JP2007522800A (ja) 2007-08-16
KR20060122927A (ko) 2006-11-30
EP1711630A2 (fr) 2006-10-18
CA2551677A1 (fr) 2005-07-28
RU2006129631A (ru) 2008-02-27

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